A second E. coli mutant lacking 4-thiouridine (s4U) in its tRNA has been identified. The absolute deficiency arises from the lack of the first of two thiolating enzymes. The pattern of the other tRNA thionucleotides is normal. Some s4U-free tRNA species have been purified for further use. The unidentified thiopurine produced after bacteriophage lambda infection of E. coli occurs in a species of tRNA overproduced on induction. This tRNA species thus far accepts no amino acid. Two different amber mutations in the thiolation region of lambda are relieved by tyrosine- but not by glutamic acid- or serine-inserting suppressors.